The continuing increase in the number of surgery, transplantation, cancer and other immunocompromized patients, that need treatment for fungal infections, together with the fact that only one new class of antifungal therapeutics has been introduced to the market in over 30 years has created an immediate need for new and better antifungal drugs with novel modes of action (MoA). The natural product compound Aureobasidin A (AbA) is a potent, fungicidal drug with a novel MoA that also does not elicit resistant pathogen strains. Unfortunately, although efficacious and very well tolerated, native AbA's target spectrum is too narrow to be clinically attractive. Of the two major human pathogens, Candida spp. and Aspergillus spp., AbA only has efficacy against Candida. However, exploratory synthetic chemistry work has demonstrated that structural modifications can convert native AbA into compounds that have close to equal efficacy against both pathogens. The required chemistry, however, is complicated and expensive, to the extent that it constitutes a barrier against development of these compounds into commercial products. The overall goal of the project outlined in this proposal is to use a novel genetic engineering approach to introduce the structural modifications required to confer Aspergillus spp. activity to AbA, thereby avoiding the high cost of synthetic chemistry and allow commercialization of an efficacious, well tolerated antifungal drug with a novel MoA. In Phase I, the gene, aba 1, encoding the non-ribosomal peptide synthetase (NRPS) complex responsible for synthesis of AbA in the producer organism was identified, cloned, sequenced and mapped. Phase II has to date produced methodologies and a set of genetic tools that allow efficient engineering of the aba 1 gene. Also accomplished to date is the successful engineering of the aba 1 gene, the generation of engineered strains producing structurally modified AbA molecules and the generation of significant new data on the unique properties of fungal NRPS complexes. Production of structurally altered cyclic peptides by engineering of a fungal NRPS complex has not been reported previously. The project has to date produced two publications, one issued patent and one pending patent application. The continued Phase II work will involve engineering of the specific modifications required to confer Aspergillus spp. activity to AbA and the preparation/selection of a producer strain capable of high production levels. Successful completion of the project will: [1] provide an efficient, well-tolerated drug to a market with a strong demand for new products;[2] address a very immediate need from a growing patient population which currently have very few treatment options;and [3] provide proof of concept and critical tools for a novel and potentially very powerful approach to the discovery of new and improved therapeutics. PUBLIC HEALTH RELEVANCE: The continuing increase in the number of surgery, transplantation, cancer and other immunocompromized patients, that need treatment for fungal infections, has generated an immediate unmet need for new antifungal drugs with novel modes of action. The proposed project will add a potent, efficacious, well-tolerated and economical drug to an inventory of antifungal drugs that currently is both limited and associated with significant limitations.